Models for stem cell differentiation leading to endothelial and hematopoietic cells are of interest because of the clinical value of stem cells and their progeny. A hallmark of stem and progenitor cells is their ability to proliferate and give rise to functional progeny, and progenitor cells are identified by their clonogenic potential. Methods previously used do not guarantee that single endothelial cells have been isolated and characterized to identify the progenitors.
Endothelial cell proliferation in vivo in normal, mature arterial, venous, and capillary vessels in most mammals is reported to be extremely low, if not nonexistent. In some experimental animals, such as pigs and dogs, radiolabeling studies have demonstrated 0.6-3.0% endothelial cell turnover daily with the dividing cells restricted to focal areas in certain vessels. Whether these dividing endothelial cells are unique and possess proliferative potential that is lacking in other mature endothelium remains undetermined.
In marked contrast, plating of endothelial cells derived from human or animal vessels in vitro is associated initially with brisk endothelial cell proliferation. For example, human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) are two commonly studied models for in vitro analysis of endothelial cell functions. Both HUVEC and BAEC cells proliferate well initially in culture but cell division wanes with time and cells become senescent and fail to divide after 15-20 passages. It is unknown if each endothelial cell derived from the vessels possesses similar proliferative potential or if only some of the cells can divide.
Angiogenesis (neoangiogenesis) is the process of new vessel formation from pre-existing vessels; this is the process reported to give rise to new vessels in adult subjects. Recently, bone marrow derived circulating endothelial progenitor cells (EPCs) have been described and these cells have also been reported to play a role in new vessel formation, at least in some experimental murine ischemic or tumor models. Conflicting evidence indicates that bone marrow derived (EPCs) do not contribute to the endothelial lining of normal arterial, venous, and capillary vessels during development and play only a minor role in neoangiogenesis. A relationship between circulating EPCs and the endothelial cells with proliferative potential that reside in normal vessels is unknown.
Emerging evidence to support the use of EPCs for angiogenic therapies or as biomarkers to assess a patient's cardiovascular disease risk and progression is accumulating and is generating enthusiasm. However, there is no uniform definition of an EPC, which makes interpretation of these studies problematic and prohibits reproduction of cell types suitable for clinical use. Although a hallmark of stem and progenitor cells (e.g. hematopoietic, intestinal, neuronal) is their ability to proliferate and give rise to functional progeny, EPCs are primarily defined by the expression of selected cell surface antigens. Sole dependence on cell surface expression of molecules can be problematic because the expression may vary with the physiologic state of the cell. No assay is reported to assess the proliferative potential (an intrinsic response) in individual endothelial cells or EPCs and thus, no comparative analysis is available.
Previous studies reported that populations of cells termed “endothelial progenitor cells” can be isolated from human umbilical cord blood or adult peripheral blood by culturing either sorted cells expressing the cell surface antigen CD34, or mononuclear cells in defined culture conditions.
Hematopoietic and endothelial progenitor cells share a number of cell surface markers in the developing yolk sac and embryo, and genetic disruption of numerous genes affects both hematopoietic and endothelial cell development. Therefore, these lineages are hypothesized to originate from a common precursor, the hemangioblast. A hierarchy of stem and progenitor cells in hematopoietic cell development is reported. Hematopoietic progenitor cells within the hierarchy are identified by their clonogenic and proliferative potential. Although genetic studies clearly show that the origin of endothelial cells is closely linked to hematopoietic cell development, evidence to support a similar hierarchy of stem and progenitor endothelial cells based on differences in proliferative potential has not been established. That is, a hierarchy of EPCs that can be discriminated by the clonogenic and proliferative potential of individual cells analogous to the hematopoietic cell system has not been reported.
Both hematopoietic stem and progenitor cells (HSC/Ps) are enriched in umbilical cord compared to adult peripheral blood. Cord blood is currently used as an alternative resource of hematopoietic stem cells for transplantation of patients with a variety of hematological disorders and malignancies.
Thousands of patients require a hematopoietic stem cell (HSC) transplant each year. Nearly ⅔ of the patients are unable to find a human leukocyte antigen (HLA) compatible match for the transplant. This is particularly true for many ethnic populations and under-represented minorities. Only ⅓ of Caucasian patients find suitable matched sibling grafts—the most compatible source with the least graft versus host disease (GVHD) complications.
Human umbilical cord blood is known to be an alternative source of HSCs for clinical transplantation. Whether or not the donor cord blood is a full major histocompatible match to the recipient or is mismatched, cord blood cells engraft and repopulate conditioned hosts as a treatment for a variety of congenital or acquired hematologic disorders. Even if the cord blood graft is mismatched with the recipient by two or more loci, the incidence and severity of GVHD is significantly less than that observed for transplantation of a similarly mismatched adult marrow or mobilized peripheral blood graft.
Limitations to a more widespread use of cord blood for transplant include the fact that only a limited number of HSC and progenitor cells are present in a graft. Because most patients do not have a matched sibling donor, most cord blood grafts are transplanted into mismatched recipients. Multiple studies report that the dose of cord blood cells in a graft is critical for patient survival when the graft comes from an unrelated donor. Transplant related mortality is reported as 20% in recipients that obtained a cord blood graft with >1.7×105 CD34+ cells/kg versus 75% in those receiving fewer CD34+ cells in the graft. Finding a method to effectively expand cord blood HSC ex vivo to increase the number of cells in a graft, would be a major advance for clinical transplantation and would have a significant commercial market.
Approaches to cord blood HSC expansion have not been impressive. In most studies, addition of a variety of growth factors to cord blood mononuclear cells or isolated CD34+ cells has been correlated with increases in total cell numbers, colony forming unit cell (CFC) numbers, and in short-term progenitor cell engraftment in immunodeficient (NOD/SCID) mice or fetal sheep. However, few approaches have been effective in increasing the number of HSC as measured by SCID repopulating cells (SRC) frequency in NOD/SCID mice or long-term engraftment in fetal sheep. The results of using expanded cord blood HSC in human patients have been disappointing.